Prostate--a novel target for evaluation of FSH modulating peptides

Effects of prostatic inhibin peptide and its synthetic fragments on FSH biosynthesis by the human pituitary and prostate, were examined in vitro. The results showed that FSH biosynthesis by prostatic tissue is modulated by these peptides in a similar fashion to that observed at the pituitary level.     

Identification of a pH sensor in the furin propeptide that regulates enzyme activation

The folding and activation of furin occur through two pH- and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg107/ generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69HRGVTKR75/, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg107. Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2- and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.     

Comprehensive splice-site analysis using comparative genomics

We have collected over half a million splice sites from five species-Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana-and classified them into four subtypes: U2-type GT-AG and GC-AG and U12-type GT-AG and AT-AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT-AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3' splice sites (3'ss) and (iv) distinct evolutionary histories of 5' and 3'ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing.     

A role for SSRP1 in recombination‐mediated DNA damage response

A possible role for structure‐specific recognition protein 1 (SSRP1) in replication‐associated repair processes has previously been suggested based on its interaction with several DNA repair factors and the replication defects observed in SSRP1 mutants. In this study, we investigated the potential role of SSRP1 in association with DNA repair mediated by homologous recombination (HR), one of the pathways involved in repairing replication‐associated DNA damage, in mammalian cells. Surprisingly, over‐expression of SSRP1 reduced the number of hprt⁺ recombinants generated via HR both spontaneously and upon hydroxyurea (HU) treatment, whereas knockdown of SSRP1 resulted in an increase of HR events in response to DNA double‐strand break formation. In correlation, we found that the depletion of SSRP1 in HU‐treated human cells elevated the number of Rad51 and H2AX foci, while over‐expression of the wild‐type SSRP1 markedly reduced HU‐induced Rad51 foci formation. We also found that SSRP1 physically interacts with a key HR repair protein, Rad54 both in vitro and in vivo. Further, branch migration studies demonstrated that SSRP1 inhibits Rad54‐promoted branch migration of Holliday junctions in vitro. Taken together, our data suggest a functional role for SSRP1 in spontaneous and replication‐associated DNA damage response by suppressing avoidable HR repair events. J. Cell. Biochem. 108: 508–518, 2009. © 2009 Wiley‐Liss, Inc.     

Heightened NTPDase-1/CD39 expression and angiogenesis in radiation proctitis

Radiation proctitis is an inflammatory process associated with persistent and refractory lower gastrointestinal bleeding. Purinergic signaling regulates hemostasis, inflammation, and angiogenesis. For example, CD39, the vascular ectonucleotidase, blocks platelet activation and is required for angiogenesis. Whether CD39 expression is affected by radiation injury is unknown. The aim of this work was to study CD39 expression patterns after clinical radiation injury to the rectum. We prospectively enrolled eight patients with radiation proctitis and five gender-matched controls. Biopsies were taken from normal-appearing rectal mucosa of controls and from the normal sigmoid and abnormal rectum of patients. Expression patterns of CD39, P2Y2 receptor, CD31, CD61 integrin, and vascular endothelial growth factor receptor 2 were examined by immunostaining; levels of CD39 were further evaluated by Western blots. Chronic inflammatory lesions of radiation proctitis were associated with heightened levels of angiogenesis. Immunohistochemical stains showed increased vascular expression of CD39, as confirmed by Western blots. CD39 was co-localized with vascular endothelial markers CD31 and CD61 integrin, as well as expressed by stromal tissues. Development of neovasculature and associated CD39 expression in radiation proctitis may be associated with the chronic, refractory bleeding observed in this condition.     

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB_69-333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB_69-333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB_69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.